1. Field of the Invention
The invention is directed to novel mutant cellobiohydrolases and cellobiohydrolase comprising compositions which have improved CBHI activity. Specifically, the present invention relates to a family of cellobiohydrolases from fungi and bacteria which are related to CBH-I produced by Talaromyces emersonii, which have certain mutations.
2. Description of Related Art
Cellulases are enzymes which are capable of hydrolysis of the beta-D-glucosidic linkages in celluloses. Cellulolytic enzymes have been traditionally divided into three major classes: endoglucanases, exoglucanases or cellobiohydrolases and beta.-glucosidases (Knowles, J. et al., (1987), TIBTECH 5, 255-261); and are known to be produced by a large number of bacteria, yeasts and fungi.
Primary among the applications that have been developed for the use of cellulolytic enzymes are those involving degrading (wood) cellulose pulp into sugars for (bio)ethanol production, textile treatments like ‘stone washing’ and ‘biopolishing’, and in detergent compositions.
Thus, cellulases have been shown to be effective in many industrial processes. Accordingly, there has been a trend in the field to search for specific cellulase compositions or components which have particularly effective performance profiles with respect to one or more specific applications. In this light, cellulases produced (expressed) in fungi and bacteria have been subject of attention. For example, cellulase produced by certain fungi such as Trichoderma spp. (especially Trichoderma longibrachiatum) have been given much attention because a complete cellulase system capable of degrading crystalline forms of cellulose is readily produced in large quantities via fermentation procedures. This specific cellulase complex has been extensively analyzed to determine the nature of its specific components and the ability of those components to perform in industrial processes. For example, Wood et al., “Methods in Enzymology”, 160, 25, pages 234 et seq. (1988), disclose that complete fungal cellulase systems comprise several different enzyme classifications including those identified as exo-cellobiohydrolases (EC 3.2.1.91) (“CBH”), endoglucanases (EC 3.2.1.4) (“EG”), and beta-glucosidases (EC 3.2.1.21) (“BG”). The fungal cellulase classifications of CBH, EG and BG can be further expanded to include multiple components within each classification. Some genetic modification of CBH-I has been proposed. S. P. Voutilainen, P. G. Murray, M. G. Tuohy and A. Koivula, Protein Engineering, Design and Selection, pp. 1-11, 2009, discloses the expression of Talaromyces emersonii cellobiohydrolase CEL7A in Saccharomyces cerevisiae and rational mutagenisis to improve its thermostability and activity. In this disclosure the mutant N54C/P191C showed increased thermostability and improved activity kcat 35.9 min-1 (table II). However this activity is still relatively low.
WO2010/122141 discloses a CBH-I from Talararomyces emersonii and polynucleotides encoding the CBH-I, and cells that incorporate these polynucleotides. The amino acid sequence of the CBH-I of WO2010/122141 is herein given as SEQ ID NO: 1.
Despite knowledge in the art related to many cellulase compositions having applications in some or all of the above areas, there is a continued need for new cellulase compositions which have improved activity under conditions of lignocellulose conversion. Therefore there is a need to improve the existing CBH-I activity, alone or in combination with other cellulases.